CN101570784B - Signal combination coding-based DNA ligation sequencing method - Google Patents
Signal combination coding-based DNA ligation sequencing method Download PDFInfo
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- CN101570784B CN101570784B CN2009100268921A CN200910026892A CN101570784B CN 101570784 B CN101570784 B CN 101570784B CN 2009100268921 A CN2009100268921 A CN 2009100268921A CN 200910026892 A CN200910026892 A CN 200910026892A CN 101570784 B CN101570784 B CN 101570784B
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Abstract
The invention discloses a signal combination coding-based DNA ligation sequencing method, which relates to a DNA ligation sequencing method using signal combination coding and belongs to the technicalfield of biology. The method is characterized by connecting signal codes in a ligation sequencing reaction, realizing the sequencing process of synchronously detecting base types in a number which isa power of the number of markers in one ligation reaction by using the same number of markers, exponentially reducing sequencing time, considerably improving sequencing throughput and reducing sequencing cost. The method uses the combinations of the signal statuses in the detection of the prior a plurality of markers for coding, makes the combinations correspond to the types of detected bases oneby one and improves the resolution power for markers in the same number due to the fact that the number of the combinations of the signal statuses of the plurality of markers exponentially grows withthe increase of the number of the types of the markers, thereby detecting the base types in the number which is the power of the number of the markers in one sequencing reaction.
Description
Technical field
The present invention is based on signal combination coded DNA connection sequence measurement and relate to a kind of employing signal combination coded DNA connection sequence measurement, is a kind of method that realizes dna sequence analysis, belongs to biological technical field.
Technical background
Along with finishing of going deep into of genome research, the particularly Human Genome Project and various model animals genome plans, biological study and medical research mode have produced dramatic change.From the difference of gene level understanding life, disease takes place, the rule of development, the interaction of medicine and life entity, and the inner interindividual hereditary difference of hereditary difference between the different plant species and same species becomes possibility.Although the factor that causes disease to take place is numerous, gene order difference (comprising single nucleotide polymorphism, dna methylation etc.) is widely regarded as an important internal factor.Most complex diseases take place and development, as cancer, diabetes, cardiovascular disorder, mental disorder etc., are the coefficient results of numerous genes and environment.By to the extensive detection of transgenation in a large amount of genome samples of a certain specified disease and with the comparison of non-ill crt gene group sample, can obtain the genotype information relevant with this disease, to the examination in drug sensitive gene mutational site, can obtain that clinical treatment and medication are had the information of guiding significance by subsequently.No matter be hereditary difference between the nearer species of sibship, the hereditary difference between the inner Different Individual of still same species all mainly is that the form with gene order difference embodies.Therefore, how the gene order difference in the quick screening genome becomes one of major subjects of genome times afterwards comprehensively.
The detection method of existing gene order difference is mainly: traditional Sanger dna sequencing method, restriction enzyme digestion length polymorphism, single strand conformation polymorphism and based on oligonucleotide probe hybridization method of gene chip etc.In these methods, only there is traditional Sanger dna sequencing method can finish comprehensive sequencing to target fragment, all the other methods all can only be determined a part of sequence information seldom.But there are deficiencies such as flux is low, cost is high, length consuming time in traditional Sanger dna sequencing method.The expense that first human genomic sequence is measured is approximately 1,000,000,000 dollars.Although this expense has been reduced to 2,000 ten thousand dollars at present, still be the bottleneck of restriction functional genome research, the cost that reduces dna sequencing significantly will promote development of life science greatly.For this reason, U.S. Venter foundation proposed the goal in research of 1000 dollars of human genome sequencings in 2003.At the beginning of 2004, the U.S. drops into state-run commune hospital more than 4,000 ten thousand dollars and supports the plan of dna sequencing Research on New, and accumulative total is above 100,000,000 dollars.Its target is the human complete genome DNA sequencing technologies of 100,000 dollars of development, and final the attenuating is 1000 dollars.
At present, except that to the existing improvement based on electrophoretic sequencing technologies, the sequencing technologies of researching and developing can be divided into four classes generally.The first kind is to extend sequencing, the base of signal mark is just joined detect in the DNA of polymerization reaction take place chain, and second class is to connect sequencing, the oligonucleotide fragment of signal mark is joined in the DNA chain that ligation takes place detect; The 3rd class is a Sequencing by hybridization, by preparing the hybridization signal of one group of high density oligonucleotide micro-array chip, carries out the Sequence Identification of target gene; The 4th class is a series of technology that can check order on monomolecular level such as molecular image; Last class technology is that the inducing DNA molecule wriggles by very trickle aperture, by electronics or method of optics base is read in this process, also becomes the nano pore sequencing technologies.Have only at present and extend sequence measurement and be connected sequence measurement and be used for genome sequencing, and developed commercial instrument and reagent, improved the efficient of dna sequencing greatly, reduced the cost of dna sequencing significantly.Yet the dna sequencing of a new generation still can not satisfy the needs of life science at aspects such as order-checking cost, flux and speed at present.One of the main reasons be single in the signal marking method that detects nucleic acid, efficient is not high.For example, connect in the sequence measurement at DNA, be subjected to the restriction of marker (as fluorophor) kind, each ligation generally only can be determined the information of a base, determine two and two above base information as need a ligation, the marker kind that then needs presents exponential growth.Therefore, effectively improve the signal marking method of nucleic acid, will increase substantially speed, the attenuating reagent cost of dna sequencing.
Summary of the invention
Purpose of the present invention is exactly at the connection sequencing technologies that has DNA now is single in the signal marking method that detects nucleic acid, efficient is not high, attempt to carry out two dimension or multidimensional coding by the combination of different marker states, composite signal coded DNA order-checking probe, thereby the more base of markers tests of equal amts or the purpose of base combination are used in realization, set up fast, accurate, low-cost and high-throughout sequencing method.
DNA connects order-checking and belongs to the synthetic sequence measurement of a kind of DNA.The basic step that DNA connects order-checking is: the sequencing primer of one of hybridization and specific region complementary pairing on dna profiling to be measured at first; Then add a kind of or one group of oligonucleotide sequencing probe in the reactor that contains template to be measured and sequencing primer, each oligonucleotide probe is made up of three parts: degeneracy base or non-strict pairing base, marker and order-checking base; Degeneracy base or non-strict pairing base are one group of nonspecific can combinations with the base of sequencing template hybridization, and marker is used for labeled oligonucleotide probe, is convenient to detect after generation DNA ligation; The order-checking base is one and a plurality of definite bases, be used for making the order-checking probe selectively with part sequencing template-sequencing primer mixture generation ligation, the order-checking base complementrity pairing of the corresponding base of the sequencing template that reacts and order-checking probe, simultaneously, there are certain corresponding relation in order-checking base and marker; After having carried out DNA connection sequencing reaction, utilize the relevant detection means that sequencing template-primer-probe complex is detected, obtain corresponding signal information, utilize the base information of corresponding position in the corresponding relation interpretation sequencing template of predefined order-checking base and marker again.
When finish once connect sequencing reaction after, remove the marker on sequencing template-primer-probe complex, carry out next time connection sequencing reaction with the order-checking probe as new connection source, carry out ligation continuously until finishing required order-checking length.Sequencing primer separates with template sex change to be measured, and the sequencing primer of of hybridization and the translation of article one primer sequence and a plurality of bases on template to be measured carries out repeatedly ligation of a new round subsequently; Repetition sex change, hybridization, connection procedure are all finished mensuration until the base information of all positions after finishing.
Technical solution of the present invention: the present invention is a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that at a certain specific dna sequencing probe, utilize the row labels that is combined into of multiple marker state, for a collection of dna sequencing probe, adopt the various combination scheme of multiple marker state to carry out mark, preparation one cover signal combination coded DNA order-checking probe, thus be implemented in when detecting differentiation and discriminating to different dna sequencing probes:
The preparation of A one cover signal combination coded DNA order-checking probe: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition.The order-checking base is used for measuring by base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any one in A, T, four kinds of bases of C, G.After finishing the preparation of unlabelled dna sequencing probe, at each unlabelled dna sequencing probe, adopt a kind of row labels that is combined into of multiple marker state, every kind of marker mark on this dna sequencing probe whether reach the amount of mark by of the combination of this marker at state in pairing state decision.Adopt different combinations of states schemes that different unmarked dna sequencing probes is carried out mark, finish the preparation of a cover signal combination coded DNA order-checking probe.
B utilizes the order-checking flow process of an above-mentioned cover signal combination coded DNA order-checking probe as follows:
A. choosing a sequencing primer and dna profiling to be measured hybridizes according to the base complementrity pairing mechanism;
B. in reaction system, add above-mentioned cover signal combination coded DNA order-checking probe and a dna ligase and a reaction system thereof, carry out the DNA ligation;
C. after finishing the DNA ligation, detect the signal of the marker of whole participant status combinations, every kind of residing state of marker signal of interpretation, combined situation according to whole marker states, determine the kind of the dna sequencing probe of generation ligation, thereby determine the type of order-checking base on this order-checking probe and arrange, and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after finishing detection, remove the marker on the dna sequencing probe to the marker signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, determined until the full sequence information in the zone to be measured of whole unknown dna profilings.
Described multiple marker carries out composite marking to unmarked dna sequencing probe, its scheme is with one or more markers of tense marker to same dna sequencing probe molecule, perhaps use the differing molecular of different marker marks respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
The combinations of states of described marker is " signal is arranged ", the combination of " no signal " two states of marker, or the combination of marker unlike signal intensity state.
Described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings, one group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can finish the examining order to the dna profiling total length in number wheel sequencing reaction; Universal sequence on the sequencing template is to add by ligation in the sequencing template preparation process, perhaps introduces by primer in amplification procedure.
Described dna profiling is to increase the segmental amount of interested purpose in the genome by dna fragmentation to be measured is passed through the DNA cloning technology, and amplification is a substance, the purpose fragment that promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base by chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
Described marker is the fluorophor that adopts, or utilizes the change of order-checking probe character chemistry, physics, changes as resistance change, electric current.
Described detection is meant with described marker character and adapts, when the fluorescent mark group of laser excitation, laser excitation intensity can be a plurality of varying strengths such as 95%, 70%, 45%, and photomultiplier transit can be respectively between a plurality of detection zones such as 95%, 70%, 45%.
Removing of described marker is that labelling groups passes through method and dna sequencing probe separates chemistry, physics, and character perhaps dna sequencing probe chemistry, physics is recovered original state.Removing of marker is only to remove marker itself, perhaps removes simultaneously with marker to link to each other or disjunct one or more base and relevant group.
Described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
Know-why: the present invention is a kind of based on signal combination coded DNA connection sequence measurement, and its know-why can be expressed as follows:
If the existing different signal tracer of n kind, every kind of order-checking probe carries out mark by the assembled state of n kind signal tracer.The assembled state here can be simple " signal is arranged ", " no signal " two-dimensional state, also can be multidimensional states such as " 100% signal ", " 75% signal ", " 50% signal ", " 25% signal ", " 0% signal ".Here we are example with the state of two dimension, utilize each marker " signal is arranged ", " no signal " two states when detected to carry out binary coding, and we are recorded as " 1 " to " signal is arranged " here, and " no signal " is recorded as " 0 ".When adopting two kinds of markers for a certain specific order-checking probe, two groups " signal is arranged " and the combination of " no signal " state by these two kinds of markers generations, obtain 2 * 2=4 kind assembled state altogether, i.e. " marker 1 has signal; marker 2 has signal ", " marker 1 has signal; marker 2 no signals ", " marker 1 no signal; marker 2 has signal ", " marker 1 no signal; marker 2 no signals ", four kinds of assembled state are recorded as bit " 11 " respectively, " 10 ", " 01 ", " 00 " (high position of dibit writes down the state of 1 labelled notation thing, low level writes down the state of 2 labelled notation things), thus realized adopting two kinds of markers with four kinds of order-checking probes of tense marker (accompanying drawing 1).When marker quantity is n (n is the integer more than or equal to 2), each assembled state of record n kind marker is a n bit, and each of bit writes down a kind of detected state of marker, and " 1 " expression detects signal, " 0 " expression does not detect signal, whole 2
n2 of individual n bit and n signal tracer
nIt is corresponding one by one to plant assembled state, and n kind marker altogether can be with tense marker 2
nPlant the order-checking probe.
Four kinds of assembled state of two kinds of markers of table 1 detect the Verbose Listing of order-checking probe type
(" 1 " expression detects signal in the state, and " 0 " expression does not detect signal)
| Order-checking probe type to be measured | Marker 1 | |
| The base Class1 | 1 | 1 |
| |
1 | 0 |
| |
0 | 1 |
| |
0 | 0 |
Have 4 kinds owing to constitute the common base of DNA, be respectively VITAMIN B4 (A), guanine (G), thymus pyrimidine (T), cytosine(Cyt) (C), 4 types of order-checking probes that therefore need accurately to detect a base need two kinds of markers " signal to be arranged " and 4 kinds of assembled state of " no signal " (seeing Table 1); The order-checking probe combinations of 2 based compositions has 4 * 4=16 type, and 4 kinds of markers of needs totally 2 * 2 * 2 * 2=16 kind assembled state detect; The rest may be inferred, and the base combination of n (n for more than or equal to 2 integer) based composition needs 2n kind marker totally 2 altogether
2nKind of composite signal state detects, and every kind of combined state comes unique expression by a 2n bit, and with the type corresponding one by one (seeing Table 2) of order-checking probe.
Table 22n kind marker 2
2nPlant the Verbose Listing of n base of combined state detection
(" 1 " represents that this kind marker modifies this kind order-checking probe in the table, and " 0 " represents that this kind marker do not modify this kind order-checking probe)
| The order-checking probe type | Marker 1 | |
|
…… | Marker 2n-2 | Marker 2n-1 | Marker 2n |
| Order- |
1 | 1 | 1 | …… | 1 | 1 | 1 |
| Order- |
1 | 1 | 1 | …… | 1 | 1 | 0 |
| Order- |
1 | 1 | 1 | …… | 1 | 0 | 1 |
| …… | …… | …… | …… | …… | …… | …… | …… |
| Order-checking probe 2 2n-2 | 0 | 0 | 0 | 0 | 1 | 0 | |
| Order-checking probe 2 2n-1 | 0 | 0 | 0 | …… | 0 | 0 | 1 |
| Order- |
0 | 0 | 0 | …… | 0 | 0 | 0 |
Beneficial effect of the present invention:
1. biggest advantage of the present invention is the composite signal coding of the marker that passes through, is implemented in and detects more base or base combination in the ligation simultaneously.The present invention measures 1 base in the single ligation classification only needs 2 kinds of markers, and traditional method needs 4 kinds of markers; The present invention measures 2 bases in the single ligation classification only needs 4 kinds of markers, and traditional method needs 16 kinds of markers.
2. compare the decline that presents exponential type with traditional method owing in the single ligation, detect the required marker kind of classification of a plurality of bases, make the classification that in the single ligation, detects a plurality of bases simultaneously become possibility, shortened at double full sequence is measured the required time.
3. encode by signal combination, the minimizing of the ligation number of times that the sequence of detection equal length is required, the cost of order-checking also reduces at double.Simultaneously, by the signal combination coding, the required marker kind of base type that detects equal amts significantly reduces, and has reduced the demand to certification mark thing equipment, thereby has reduced cost.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further specified.
Fig. 1 is when two dimension when the marker state reduction, and promptly during the having or not of signal, the two-dimensional state combine detection by two kinds of markers is at the schematic diagram that once connects A in the sequencing reaction, T, C, four kinds of bases of G.1, when base to be measured was A, the order-checking base was that the assembled state of the pairing marker of order-checking probe of U is " marker 1 has signal, and marker 2 has signal ", i.e. " 1,1 "; 2, when base to be measured was C, the order-checking base was that the assembled state of the pairing marker of order-checking probe of G is " marker 1 has signal, marker 2 no signals ", i.e. " 1,0 "; 3, when base to be measured was G, the order-checking base was that the assembled state of the pairing marker of order-checking probe of C is " marker 1 no signal, marker 2 has signal ", i.e. " 0,1 "; 4, when base to be measured was T, the order-checking base was that the assembled state of the pairing marker of order-checking probe of A is " marker 1 no signal, marker 2 no signals ", i.e. " 0,0 ".
Fig. 2 is the co-operation schema of example 2, example 3 and example 4.1, the PCR product behind P1, the P2 primer amplification is fixed in surface of glass slide; 2, article one sequencing primer I1 and the PCR product hybridization of being fixed in surface of glass slide; 3, connected the oligonucleotide probe of order-checking base and PCR locations complementary to be measured on the sequencing primer I1; 4, remove the fluorescence modification group after finishing detection; 5, I1 connects the connection of the probe that carries out checking order for the second time; 6, remove the fluorescence modification group once more after finishing detection; 7, I1 finishes whole 4 ligations; 8, sequencing primer I1 separates with the sex change of PCR product; 9, second sequencing primer I2 and PCR product hybridization beginning new round sequencing reaction.
Fig. 3 is the detailed diagram that example 2 sequencing primer I1 carry out preceding twice order-checking ligation.1, sequencing primer I1 and the PCR product hybridization of being fixed in surface of glass slide, 5 ' preceding 11 bases of end and the PCR primer P2 complementary strand complementary pairing of I1,3 ' terminal 1,2,3, No. 4 base complementrity pairing of last four degeneracy base N of the 3 ' end of I1 and DNA to be measured; 2, carry out the ligation first time, the order-checking base is connected with the I1 of hybridizing on the PCR product for " 5 '-AG-3 ' " several oligonucleotide monomers (all the other positions of oligonucleotide probe are degeneracy base N) in the mixture that 32 kinds of oligonucleotide probes are formed, by detecting, can learn that connect going up the oligonucleotide probe end is marked with CY3, TXR, FTC group respectively, look into 3 ' terminal the 7th, No. 8 base that the oligonucleotide probe formula table learns DNA to be measured and be " 5 '-CT-3 ' "; 3, remove the fluorescence modification group of oligonucleotide probe end; 4, carry out connecting the second time, detect 3 ' terminal the 15th, No. 16 base of DNA to be measured.
Embodiment
Embodiment of the present invention are divided into two kinds:
First kind is at a certain order-checking probe, selects for use some kinds of markers simultaneously same order-checking probe molecule to be carried out mark.At a ligation, each marker presents the two states of " signal is arranged ", " no signal " respectively when detected, by the specific order-checking probe decision that adds in order-checking ligation system, the order-checking probe that preparation contains different markers is a key of the present invention.Add order-checking probe in the reaction system by a group echo oligonucleotide probe of different markers form.Each oligonucleotide probe is by one or more order-checking bases (measuring the base information of corresponding position among the DNA to be measured by base complementrity pairing rule), one or more degeneracy base N (N is any one in A, T, four kinds of bases of C, G) or non-strict pairing base (as xanthoglobulin etc.) and a kind of marker formation with detected ability.Combination between order-checking base and the marker is according to the listed state preparation of table 2, when the state of determined in the table 2 " order-checking probe type i " corresponding k kind marker is " 1 ", then when preparation order-checking base was the order-checking probe molecule of " base composite type i ", " order-checking probe type i " all states were the marker of " 1 " in the flag table simultaneously; Synthetic line by line table 2 listed whole 2
2nPlant oligonucleotide probe, this probe mixture is the oligonucleotide probe mixture of required preparation.
Second kind is that this kind order-checking probe is divided into several portions, and each part is a kind of marker of mark respectively.At a ligation, each marker presents the two states of " signal is arranged ", " no signal " respectively when detected, by the specific order-checking probe decision that adds in order-checking ligation system.Therefore preparing the order-checking probe that contains different markers is key of the present invention.Add order-checking probe in the reaction system by a group echo oligonucleotide probe of different markers form.Each oligonucleotide probe is by one or more order-checking bases (measuring the base information of corresponding position among the DNA to be measured by base complementrity pairing rule), one or more degeneracy base N (N is any one in A, T, four kinds of bases of C, G) or non-strict pairing base (as xanthoglobulin etc.) and a kind of marker formation with detected ability.Combination between order-checking base and the marker is according to the listed state preparation of table 2, as determined in the table 2 " base composite type i " and " marker j " when listed state be " 1 ", then preparation order-checking base is that " base composite type i ", marker are the oligonucleotide probe of " marker j "; Then not synthetic when being " 0 " as listed state, the oligonucleotide probe that the synthetic listed whole states of table 2 are " 1 ", this probe mixture is the oligonucleotide probe mixture of required preparation.
Further, the state of each marker can be defined as m kind state (m＞2) by the size of relative signal intensity.For example, the signal of marker can be " 0 ", " 1 ", " 2 " and four kinds of states such as " 3 ".Utilize each marker when detected, to have " no signal ", " 1/3 signal ", " 2/3 signal " and " full signal " four kinds of states to carry out quaternary coding, can be recorded as " 0 " to " no signal ", " 1/3 signal " is recorded as " 1 ", " 2/3 signal " is recorded as " 2 ", " full signal " is recorded as " 3 ".When adopting two kinds of markers for a certain specific order-checking probe, signal combination by these two kinds of markers generations, obtain 4 * 4=16 kind assembled state altogether, promptly (high position of two quaternary numbers writes down the state of 1 labelled notation thing " 00 ", " 01 ", " 02 ", " 03 ", " 10 ", " 11 ", " 12 ", " 13 ", " 20 ", " 21 ", " 22 ", " 23 ", " 30 ", " 31 ", " 32 ", " 33 ", low level writes down the state of 2 labelled notation things), thus realized adopting 2 kinds of markers with 16 kinds of order-checking probes of tense marker.When marker quantity is n (n is the integer more than or equal to 2), record mark thing assembled state is that whole n position quaternary number (promptly can while mark 4
nPlant the order-checking probe), each of quaternary number writes down a kind of detected state of marker, whole 4
n4 of individual n position quaternary number and n signal tracer
nIt is corresponding one by one to plant assembled state.
Embodiment 1: the present invention is a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that at a certain specific dna sequencing probe, utilize the row labels that is combined into of multiple marker state, for a collection of dna sequencing probe, adopt the various combination scheme of multiple marker state to carry out mark, preparation one cover signal combination coded DNA order-checking probe, thus be implemented in when detecting the differentiation and the discriminating of different dna sequencing probes, reach the purpose that template to be measured is checked order.
The preparation of A one cover signal combination coded DNA order-checking probe: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition.The order-checking base is used for measuring by base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any one in A, T, four kinds of bases of C, G.After finishing the preparation of unlabelled dna sequencing probe, at each unlabelled dna sequencing probe, adopt a kind of row labels that is combined into of multiple marker state, every kind of marker mark on this dna sequencing probe whether reach the amount of mark by of the combination of this marker at state in pairing state decision.Adopt different combinations of states schemes that different unmarked dna sequencing probes is carried out mark, finish the preparation of a cover signal combination coded DNA order-checking probe.
B utilizes the order-checking flow process of an above-mentioned cover signal combination coded DNA order-checking probe as follows:
A. choosing a sequencing primer and dna profiling to be measured hybridizes according to the base complementrity pairing mechanism;
B. in reaction system, add above-mentioned cover signal combination coded DNA order-checking probe and a dna ligase and a reaction system thereof, carry out the DNA ligation;
C. after finishing the DNA ligation, detect the signal of the marker of whole participant status combinations, every kind of residing state of marker signal of interpretation, combined situation according to whole marker states, determine the kind of the dna sequencing probe of generation ligation, thereby determine the type of order-checking base on this order-checking probe and arrange, and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after finishing detection, remove the marker on the dna sequencing probe to the marker signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, determined until the full sequence information in the zone to be measured of whole unknown dna profilings.
Described multiple marker carries out composite marking to unmarked dna sequencing probe, its scheme is with one or more markers of tense marker to same dna sequencing probe molecule, perhaps use the differing molecular of different marker marks respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
The combinations of states of described marker is " signal is arranged ", the combination of " no signal " two states of marker, or the combination of marker unlike signal intensity state.
Described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings, one group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can finish the examining order to the dna profiling total length in number wheel sequencing reaction; Universal sequence on the sequencing template is to add by ligation in the sequencing template preparation process, perhaps introduces by primer in amplification procedure.
Described dna profiling is to increase the segmental amount of interested purpose in the genome by dna fragmentation to be measured is passed through the DNA cloning technology, and amplification is a substance, the purpose fragment that promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base by chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
Described marker is the fluorophor that adopts, or utilizes the change of order-checking probe character chemistry, physics, changes as resistance change, electric current.
Described detection is meant with described marker character and adapts, when the fluorescent mark group of laser excitation, laser excitation intensity can be a plurality of varying strengths such as 95%, 70%, 45%, and photomultiplier transit can be respectively between a plurality of detection zones such as 95%, 70%, 45%.
Removing of described marker is that labelling groups passes through method and dna sequencing probe separates chemistry, physics, and character perhaps dna sequencing probe chemistry, physics is recovered original state.Removing of marker is only to remove marker itself, perhaps removes simultaneously with marker to link to each other or disjunct one or more base and relevant group.
Described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
2: four look fluorescence of embodiment composition coding method (respectively tagging scheme) detects on human No. 17 karyomit(e)s, 45332-45363 totally 32 base-pair sequences on the RP11-354P11 clone;
Extract human complete genome DNA, utilize PCR method amplification purpose fragment, primer is P1, P2 (sequence sees Table 3), 5 ' the terminal hydroxyl modified that adopts of primer P1.After PCR product after the amplification was fixed in aldehyde group modified surface of glass slide, heating slide to 95 ℃ made chain and fixedly chain disengaging of double-stranded PCR product, begins sequencing reaction subsequently.
Relevant oligonucleotide sequence information in table 3 example 2
| The sequence name | Sequence information |
| Sequence to be measured | 5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’ |
| P1 | OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’ |
| P2 | 5’-GCAGTTGCCAGTGTTCCAGGAGT-3’ |
| I1 | 5’-GTTCCAGGAGTNNNN-3’ |
| I2 | 5’-GTGTTCCAGGAGTNN-3’ |
| I3 | 5’-CAGTGTTCCAGGAGT-3’ |
| I4 | 5’-GCCAGTGTTCCAGGA-3’ |
Add hybridization buffer and article one sequencing primer I1 (sequence sees Table 3) to surface of glass slide, hybridized renaturation 20 minutes for 37 ℃.Add the mixture that the listed whole 32 kinds of oligonucleotide probes of table are formed in reaction tank, adopt the T4 nucleic acid ligase to carry out ligation, reaction conditions is 25 ℃ and connects 30 minutes.Utilize laser confocal microscope that slide is scanned after connection is finished, scanning adopts the wavelength corresponding to CY3, CY5, TXR and FTC to carry out respectively, according to the base information (accompanying drawing 2) of the fluorescent signal interpretation correspondence that obtains.
In the ligation first time, on I1 and DNA to be measured and the primer P2 complementary strand " 5 '-AAGAACTCCTGGAAC-3 ' " sequence hybridization, after adding the oligonucleotide probe mixture, if the order-checking base is matched with the 3rd, No. 4 base complementrity that DNA to be measured is positioned at I1 primer 3 ' end downstream in the oligonucleotide probe mixture, then these oligonucleotide probes and DNA to be measured are hybridized and with primer I 1 ligation are taken place.The the 3rd, 4 base that DNA to be measured is positioned at I1 primer 3 ' end downstream is " 5 '-CT-3 ' ", its reverse complemental base is " 5 '-AG-3 ' ", and then all order-checking bases be oligonucleotide probe " 5 '-NNAGNNNN-3 '-CY3 ", " 5 '-NNAGNNNN-3 '-CY5 ", " the 5 '-NNAGNNNN-3 '-FTC " and dna profiling to be measured and primer I 1 generation ligation (accompanying drawing 3-2) of " 5 '-AG-3 ' ".When carrying out signal detection, this takes turns ligation can detect CY3, CY5, three kinds of fluorescence of FTC simultaneously, reverse complemental chain information by 3 ' terminal the 7th, No. 8 base that can interpretation DNA to be measured of tabling look-up is " 5 '-AG-3 ' ", thereby 3 ' terminal the 7th, No. 8 base that can obtain DNA to be measured is " 5 '-CT-3 ' ".
After finishing the next round detection, utilize the fluorophor of oligonucleotide probe 3 ' end on chemical process excision link and the primer I 1, obtain a free hydroxyl at 3 ' end and carry out ligation once more (accompanying drawing 3-3).In reaction tank, add the listed whole thuja acid probe mixture of table 4 once more, carry out second and take turns ligation.Ligation this time is to detect DNA to be measured to be positioned at the 11st of I1 primer 3 ' end downstream, No. 12 base (3 ' the terminal the 15th of DNA to be measured, No. 16 bases) " 5 '-TG-3 ' ", three kinds of order-checkings this moment bases are the oligonucleotide probe " 5 '-NNCANNNN-3 '-CY3 " of " 5 '-CA-3 ' ", " 5 '-NNCANNNN-3 '-TXR ", " 5 '-NNCANNNN-3 '-FTC " the generation ligation, carrying out fluoroscopic examination is to detect CY3 simultaneously, TXR, FTC, the interpretation and carry out reverse complemental and obtain 3 ' the terminal the 15th of DNA to be measured of tabling look-up, No. 16 bases are " 5 '-TG-3 ' " (accompanying drawing 3-4).Carry out the 3rd, 4 of I1 primer once more behind the terminal fluorophor of the oligonucleotide that the chemical process excision connects and take turns ligation, record 3 ' terminal the 23rd, No. 24 base and the 31st, No. 32 base information of DNA to be measured respectively.
Table 4 oligonucleotide probe mixture Verbose Listing
After finishing the four-wheel ligation, 95 ℃ make sequencing primer I1 separate with DNA to be measured, add hybridization buffer and second sequencing primer I2 (sequence sees Table 3) to surface of glass slide, hybridize renaturation 20 minutes for 37 ℃.Begin the four-wheel ligation subsequently and detect 3 ' terminal the 5th, No. 6 of DNA to be measured respectively, 13, No. 14,21, No. 22 and 29, No. 30 four groups of bases.
Same, utilize sequencing primer I3 to detect 3 ' terminal the 3rd, No. 4 of DNA to be measured, 11, No. 12,19, No. 20 and 27, No. 28 four groups of bases, utilize sequencing primer I4 to detect 3 ' terminal the 1st, No. 2 of DNA to be measured, 9, No. 10,17, No. 18 and 25, No. 26 four groups of bases.
Splice the sequence information that can obtain whole 32 bases of DNA to be measured by the acquisition sequence information.
3: four look fluorescence of embodiment composition coding method (common tagging scheme) detects on human No. 17 karyomit(e)s, 45332-45363 totally 32 base-pair sequences on the RP11-354P11 clone;
Extract human complete genome DNA, utilize PCR method amplification purpose fragment, primer is P1, P2 (sequence sees Table 5), 5 ' the terminal hydroxyl modified that adopts of primer P1.After PCR product after the amplification was fixed in aldehyde group modified surface of glass slide, heating slide to 95 ℃ made chain and fixedly chain disengaging of double-stranded PCR product, begins sequencing reaction subsequently.
Relevant oligonucleotide sequence information in table 5 example 3
| The sequence name | Sequence information |
| Sequence to be measured | 5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’ |
| P1 | OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’ |
| P2 | 5’-GCAGTTGCCAGTGTTCCAGGAGT-3’ |
| I1 | 5’-GTTCCAGGAGTNNNN-3’ |
| I2 | 5’-GTGTTCCAGGAGTNN-3’ |
| I3 | 5’-CAGTGTTCCAGGAGT-3’ |
| I4 | 5’-GCCAGTGTTCCAGGA-3’ |
Add hybridization buffer and article one sequencing primer I1 (sequence sees Table 5) to surface of glass slide, hybridized renaturation 20 minutes for 37 ℃.In reaction tank, add the mixture that the listed whole 16 kinds of oligonucleotide probes of table 6 are formed, each oligonucleotide probe molecule according to form with tense marker 0-4 kind fluorescence molecule.Adopt the T4 nucleic acid ligase to carry out ligation, reaction conditions is 25 ℃ and connects 30 minutes.Utilize laser confocal microscope that slide is scanned after connection is finished, scanning adopts the wavelength corresponding to CY3, CY5, TXR and FTC to carry out respectively, according to the base information (accompanying drawing 2) of the fluorescent signal interpretation correspondence that obtains.
In the ligation first time, on I1 and DNA to be measured and the primer P2 complementary strand " 5 '-AAGAACTCCTGGAAC-3 ' " sequence hybridization, after adding the oligonucleotide probe mixture, if the order-checking base is matched with the 3rd, No. 4 base complementrity that DNA to be measured is positioned at I1 primer 3 ' end downstream in the oligonucleotide probe mixture, then these oligonucleotide probes and DNA to be measured are hybridized and with primer I 1 ligation are taken place.The the 3rd, 4 base that DNA to be measured is positioned at I1 primer 3 ' end downstream is " 5 '-CT-3 ' ", its reverse complemental base is " 5 '-AG-3 ' ", the base that then checks order is " 5 '-NNAGNNNN-3 ' " for the oligonucleotide probe of " 5 '-AG-3 ' ", as shown in Table 6 this oligonucleotide probe with tense marker CY3, CY5 and three kinds of fluorophors of FTC, ligation takes place with dna profiling to be measured and primer I 1.When carrying out signal detection, this takes turns ligation can detect CY3, CY5, three kinds of fluorescence of FTC simultaneously, reverse complemental chain information by 3 ' terminal the 7th, No. 8 base that can interpretation DNA to be measured of tabling look-up is " 5 '-AG-3 ' ", thereby 3 ' terminal the 7th, No. 8 base that can obtain DNA to be measured is " 5 '-CT-3 ' ".
Table 6 oligonucleotide probe mixture Verbose Listing
(a kind of oligonucleotide probe of each line display in the table, common modification group are represented to replant on each oligonucleotide probe molecule of probe and are modified the fluorophor situation simultaneously, and " having " expression is modified with this group, and " nothings " represents this group of unmodified)
After finishing the next round detection, utilize the fluorophor of oligonucleotide probe 3 ' end on chemical process excision link and the primer I 1, obtain a free hydroxyl at 3 ' end and carry out ligation once more.In reaction tank, add the listed whole thuja acid probe mixture of table 6 once more, carry out second and take turns ligation.Ligation this time is to detect DNA to be measured to be positioned at the 11st of I1 primer 3 ' end downstream, No. 12 base (3 ' the terminal the 15th of DNA to be measured, No. 16 bases) " 5 '-TG-3 ' ", the corresponding oligonucleotide probe of the base " 5 '-CA-3 ' " that checks order this moment is " 5 '-NNCANNNN-3 ' ", as shown in Table 6 this oligonucleotide probe with tense marker CY3, three kinds of fluorophors of TXR and FTC, after carrying out ligation, detect CY3 simultaneously, TXR, FTC, the interpretation and carry out reverse complemental and obtain 3 ' the terminal the 15th of DNA to be measured of tabling look-up, No. 16 bases are " 5 '-TG-3 ' ".Carry out the 3rd, 4 of I1 primer once more behind the terminal fluorophor of the oligonucleotide that the chemical process excision connects and take turns ligation, record 3 ' terminal the 23rd, No. 24 base and the 31st, No. 32 base information of DNA to be measured respectively.
After finishing the four-wheel ligation, 95 ℃ make sequencing primer I1 separate with DNA to be measured, add hybridization buffer and second sequencing primer I2 (sequence sees Table 5) to surface of glass slide, hybridize renaturation 20 minutes for 37 ℃.Begin the four-wheel ligation subsequently and detect 3 ' terminal the 5th, No. 6 of DNA to be measured respectively, 13, No. 14,21, No. 22 and 29, No. 30 four groups of bases.
Same, utilize sequencing primer I3 to detect 3 ' terminal the 3rd, No. 4 of DNA to be measured, 11, No. 12,19, No. 20 and 27, No. 28 four groups of bases, utilize sequencing primer I4 to detect 3 ' terminal the 1st, No. 2 of DNA to be measured, 9, No. 10,17, No. 18 and 25, No. 26 four groups of bases.
Splice the sequence information that can obtain whole 32 bases of DNA to be measured by the acquisition sequence information.
Embodiment 4: Two Colour Fluorescence light intensity composition coding method detects on human No. 17 karyomit(e)s, 45332-45363 totally 32 base-pair sequences on the RP11-354P11 clone;
Extract human complete genome DNA, utilize PCR method amplification purpose fragment, primer is P1, P2 (sequence sees Table 7), 5 ' the terminal hydroxyl modified that adopts of primer P1.After PCR product after the amplification was fixed in aldehyde group modified surface of glass slide, heating slide to 95 ℃ made chain and fixedly chain disengaging of double-stranded PCR product, begins sequencing reaction subsequently.
Relevant oligonucleotide sequence information in table 7 example 4
| The sequence name | Sequence information |
| Sequence to be measured | 5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’ |
| P1 | OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’ |
| P2 | 5’-GCAGTTGCCAGTGTTCCAGGAGT-3’ |
| I1 | 5’-GTTCCAGGAGTNNNN-3’ |
| I2 | 5’-GTGTTCCAGGAGTNN-3’ |
| I3 | 5’-CAGTGTTCCAGGAGT-3’ |
| I4 | 5’-GCCAGTGTTCCAGGA-3’ |
Add hybridization buffer and article one sequencing primer I1 (sequence sees Table 7) to surface of glass slide, hybridized renaturation 20 minutes for 37 ℃.Add the mixture that the listed whole 16 kinds of oligonucleotide probes of table 8 are formed in reaction tank, adopt the T4 nucleic acid ligase to carry out ligation, reaction conditions is 25 ℃ and connects 30 minutes.Utilize laser confocal microscope that slide is scanned after connection is finished, scanning adopts the wavelength corresponding to CY3, CY5 to carry out respectively, according to the base information (accompanying drawing 2) of the fluorescent signal interpretation correspondence that obtains.
In the ligation first time, on I1 and DNA to be measured and the primer P2 complementary strand " 5 '-AAGAACTCCTGGAAC-3 ' " sequence hybridization, after adding the oligonucleotide probe mixture, if the order-checking base is matched with the 3rd, No. 4 base complementrity that DNA to be measured is positioned at I1 primer 3 ' end downstream in the oligonucleotide probe mixture, then these oligonucleotide probes and DNA to be measured are hybridized and with primer I 1 ligation are taken place.The the 3rd, 4 base that DNA to be measured is positioned at I1 primer 3 ' end downstream is " 5 '-CT-3 ' ", its reverse complemental base is " 5 '-AG-3 ' ", the base that then checks order is " 5 '-NNAGNNNN-3 ' " for the oligonucleotide probe of " 5 '-AG-3 ' ", as shown in Table 8 in this oligonucleotide probe 100% mark the CY3 group, 33.3% mark the CY5 group, ligation takes place with dna profiling to be measured and primer I 1.When carrying out signal detection, this takes turns ligation can detect CY3 and two kinds of fluorescence of CY5 simultaneously, the CY3 fluorescence intensity is 100% of a normal intensity simultaneously, the CY5 fluorescence intensity is 33.3% of a normal intensity, reverse complemental chain information by 3 ' terminal the 7th, No. 8 base that can interpretation DNA to be measured of tabling look-up is " 5 '-AG-3 ' ", thereby 3 ' terminal the 7th, No. 8 base that can obtain DNA to be measured is " 5 '-CT-3 ' ".
Table 8 oligonucleotide probe mixture Verbose Listing
(a kind of oligonucleotide probe of each row representative, the CY3 ratio represents to modify in the oligonucleotide probe ratio of CY3 group, modifies the ratio of CY5 group in the CY5 ratio oligonucleotide probe)
| The order-checking base | Oligonucleotide chain | The CY3 ratio | The CY5 ratio |
| AA | 5’-NNAANNNN-3’ | 100％ | 100％ |
| AC | 5’-NNACNNNN-3’ | 100％ | 66.7％ |
| AG | 5’-NNAGNNNN-3’ | 100％ | 33.3％ |
| AT | 5’-NNATNNNN-3’ | 100％ | 0％ |
| CA | 5’-NNCANNNN-3’ | 66.7％ | 100％ |
| CC | 5’-NNCCNNNN-3’ | 66.7％ | 66.7％ |
| CG | 5’-NNCGNNNN-3’ | 66.7％ | 33.3％ |
| CT | 5’-NNCTNNNN-3’ | 66.7％ | 0％ |
| GA | 5’-NNGANNNN-3’ | 33.3％ | 100％ |
| GC | 5’-NNGCNNNN-3’ | 33.3％ | 66.7％ |
| GG | 5’-NNGGNNNN-3’ | 33.3％ | 33.3％ |
| GT | 5’-NNGTNNNN-3’ | 33.3％ | 0％ |
| TA | 5’-NNTANNNN-3’ | 0％ | 100％ |
| TC | 5’-NNTCNNNN-3’ | 0％ | 66.7％ |
| TG | 5’-NNTGNNNN-3’ | 0％ | 33.3％ |
| TT | 5’-NNTTNNNN-3’ | 0％ | 0％ |
After finishing the next round detection, utilize the fluorophor of oligonucleotide probe 3 ' end on chemical process excision link and the primer I 1, obtain a free hydroxyl at 3 ' end and carry out ligation once more.In reaction tank, add the listed whole thuja acid probe mixture of table 6 once more, carry out second and take turns ligation.Ligation this time is to detect DNA to be measured to be positioned at the 11st of I1 primer 3 ' end downstream, No. 12 base (3 ' the terminal the 15th of DNA to be measured, No. 16 bases) " 5 '-TG-3 ' ", the corresponding oligonucleotide probe of the base " 5 '-CA-3 ' " that checks order this moment is " 5 '-NNCANNNN-3 ' ", as shown in Table 8 this oligonucleotide probe 66.7% mark the CY3 group, 100% mark the CY5 group, after carrying out ligation, detect CY3 and CY5 simultaneously, and the CY3 fluorescence intensity is 66.7% of a normal intensity, the CY5 fluorescence intensity is 100% of a normal intensity, the interpretation and carry out reverse complemental and obtain 3 ' the terminal the 15th of DNA to be measured of tabling look-up, No. 16 bases are " 5 '-TG-3 ' ".Carry out the 3rd, 4 of I1 primer once more behind the terminal fluorophor of the oligonucleotide that the chemical process excision connects and take turns ligation, record 3 ' terminal the 23rd, No. 24 base and the 31st, No. 32 base information of DNA to be measured respectively.
After finishing the four-wheel ligation, 95 ℃ make sequencing primer I1 separate with DNA to be measured, add hybridization buffer and second sequencing primer I2 (sequence sees Table 7) to surface of glass slide, hybridize renaturation 20 minutes for 37 ℃.Begin the four-wheel ligation subsequently and detect 3 ' terminal the 5th, No. 6 of DNA to be measured respectively, 13, No. 14,21, No. 22 and 29, No. 30 four groups of bases.
Same, utilize sequencing primer I3 to detect 3 ' terminal the 3rd, No. 4 of DNA to be measured, 11, No. 12,19, No. 20 and 27, No. 28 four groups of bases, utilize sequencing primer I4 to detect 3 ' terminal the 1st, No. 2 of DNA to be measured, 9, No. 10,17, No. 18 and 25, No. 26 four groups of bases.
Splice the sequence information that can obtain whole 32 bases of DNA to be measured by the acquisition sequence information.
Claims (7)
1. one kind connects sequence measurement based on the signal combination coded DNA, it is characterized in that at a certain specific dna sequencing probe, utilize the row labels that is combined into of multiple marker state, for a collection of dna sequencing probe, adopt the combination of multiple marker state to carry out two dimension or multidimensional coding mark, preparation one cover signal combination coded DNA order-checking probe, thus be implemented in when detecting differentiation and discriminating to different dna sequencing probes; The state of described marker comprises that marker " has signal ", " no signal " two states, and the state of marker unlike signal intensity:
The preparation of A one cover signal combination coded DNA order-checking probe: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition; The order-checking base is used for measuring by base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any one in A, T, four kinds of bases of C, G; After finishing the preparation of unlabelled dna sequencing probe, at each unlabelled dna sequencing probe, adopt a kind of row labels that is combined into based on two dimension or multidimensional coding of multiple marker state, every kind of marker mark on this dna sequencing probe whether reach the amount of mark by of the combination of this marker at state in pairing state decision; Adopt different combinations of states schemes that different unmarked dna sequencing probes is carried out mark, finish the preparation of a cover signal combination coded DNA order-checking probe;
B utilizes the order-checking flow process of an above-mentioned cover signal combination coded DNA order-checking probe as follows:
A. choosing a sequencing primer and dna profiling to be measured hybridizes according to the base complementrity pairing mechanism;
B. in reaction system, add above-mentioned cover signal combination coded DNA order-checking probe and a dna ligase and a reaction system thereof, carry out the DNA ligation;
C. after finishing the DNA ligation, detect the signal of the marker of whole participant status combinations, every kind of residing state of marker signal of interpretation, combined situation according to whole marker states, determine the kind of the dna sequencing probe of generation ligation, thereby determine the type of order-checking base on this order-checking probe and arrange, and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after finishing detection, remove the marker on the dna sequencing probe to the marker signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, determined until the full sequence information in the zone to be measured of whole unknown dna profilings.
2. according to claim 1 a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that described multiple marker carries out composite marking to unmarked dna sequencing probe, its scheme is with one or more markers of tense marker to same dna sequencing probe molecule, perhaps use the differing molecular of different marker marks respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
3. according to claim 1 a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings, one group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can finish the examining order to the dna profiling total length in number wheel sequencing reaction; Universal sequence on the sequencing template is to add by ligation in the sequencing template preparation process, perhaps introduces by primer in amplification procedure.
4. according to claim 1 a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that described dna profiling is by dna fragmentation to be measured is increased the segmental amount of interested purpose in the genome by the DNA cloning technology, amplification is a substance, a purpose fragment promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base by chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
5. according to claim 1ly a kind ofly connect sequence measurement, it is characterized in that described marker, be the fluorophor that adopts, or utilize the change of order-checking probe character chemistry, physics based on the signal combination coded DNA.
6. according to claim 1 a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that removing of described marker, be that labelling groups passes through method and dna sequencing probe separates chemistry, physics, character perhaps dna sequencing probe chemistry, physics is recovered original state; Removing of marker is only to remove marker itself, perhaps removes simultaneously with marker to link to each other or disjunct one or more base and relevant group.
7. according to claim 1 a kind of based on signal combination coded DNA connection sequence measurement, it is characterized in that described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
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